Modified cytokines for use in cancer therapy

ABSTRACT

Cytokine derivatives capable of homing the tumoral vessels and the antigen presenting cells and the use thereof as antitumoral agents.

FIELD OF THE INVENTION

The present invention refers to modified cytokines for use in thetreatment of cancer. More particularly, the invention refers tocytokines derivatives capable of “homing” tumor vessels and antigenpresenting cells. The present invention also relates to synergisticcombinations which include a modified cytokine.

BACKGROUND OF THE INVENTION

The antitumoral activity of some cytokines is well known and described.Some cytokines have already been used therapeutically also in humans(29). For example, such cytokines as interleukine-2 (IL-2) andinterferon α(IFNα) have shown positive antitumoral activity in patientswith different types of tumors, such as kidney metastatic carcinoma,hairy cell leukemia, Kaposi sarcoma, melanoma, multiple mieloma, and thelike. Other cytokines like IFNβ, the Tumor Necrosis Factor (TNF) α,TNFβ, IL-1, 4, 6, 12, 15 and the Colony Stimulating Factors (CFSs) haveshown a certain antitumoral activity on some types of tumors andtherefore are the object of further studies.

In general, the therapeutic use of cytokines is strongly limited bytheir systemic toxicity. TNF, for example, was originally discovered forits capacity of inducing the hemorrhagic necrosis of some tumors (1),and for its in vitro cytotoxic effect on different tumoral lines (2),but it subsequently proved to have strong pro-inflammatory activity,which can, in case of overproduction conditions, dangerously affect thehuman body (3).

As the systemic toxicity is a fundamental problem with the use ofpharmacologically active amounts of cytokines in humans, novelderivatives and therapeutic strategies are now under evaluation, aimedat reducing the toxic effects of this class of biological effectorswhile keeping their therapeutic efficacy.

Some novel approaches are directed to:

-   a) the development of fusion proteins which can deliver TNF into the    tumor and increase the local concentration. For example, the fusion    proteins consisting of TNF and tumor specific-antibodies have been    produced (4);-   b) the development of TNF mutants which maintain the antitumoral    activity and have a reduced systemic toxicity. Accordingly, mutants    able of selectively recognizing only one receptor (p55 or p75) have    been already prepared (5);-   c) the use of anti-TNF antibodies able to reduce some toxic effects    of TNF without compromising its antitumoral activity. Such    antibodies have been already described in literature (30);-   d) the use of TNF derivatives with a higher half-life (for example    TNF conjugated with polyethylene glycol).

The preparation of TNF derivatives capable of selectively targeting thetumoral sites has been recently reported. For example, a fusion proteinhas been described, obtained by fusing the gene of the heavy chain of ananti-transferrin receptor mAb and the TNF gene (4), or a fusion proteinof TNF with the “hinge” region of a monoclonal antibody against thetumor-associated TAG72 antigen (6), or a Fv-TNF fusion protein (6).

EP 251 494 discloses a system for administering a diagnostic ortherapeutic agent, which comprises: an antibody conjugated with avidinor streptavidin, an agent capable of complexing the conjugated antibodyand a compound consisting of the diagnostic or therapeutic agentconjugated with biotin, which are administered sequentially andadequately delayed, so as to allow the localization of the therapeuticor diagnostic agent through the biotin-streptavidin interaction on thetarget cell recognized by the antibody. The described therapeutic ordiagnostic agents comprise metal chelates, in particular chelates ofradionuclides and low molecular weight antitumoral agents such ascis-platinum, doxorubicin, etc.

EP 496 074 discloses a method which provides the sequentialadministration of a biotinylated antibody, avidin or streptavidin and abiotinylated diagnostic or therapeutic agent. Although cytotoxic agentslike ricin are generically mentioned, the application relative toradiolabelled compounds is mostly disclosed.

WO 95/15979 discloses a method for localizing highly toxic agents oncellular targets, based on the administration of a first conjugatecomprising the specific target molecule conjugated with a ligand or ananti-ligand followed by the administration of a second conjugateconsisting of the toxic agent bound to an anti-ligand or to the ligand.

WO98/10795 discloses tumor homing molecules including peptidescontaining the amino acid sequence NGR. No use of the peptide to targeta cytokine to a tumor is described.

WO 99/13329 discloses a method for targeting a molecule to tumoralangiogenic vessels, based on the conjugation of said molecule withligands of NGR receptors. A number of molecules have been suggested aspossible candidates, but doxorubicin only is specifically described. Nouse of ligands of NGR receptors as cytokines vehicles to induce immunoresponses is disclosed.

SUMMARY OF THE INVENTION

It has now surprisingly been found that the therapeutic index of certaincytokines can be remarkably improved and their immunotherapeuticproperties can be enhanced by coupling with a ligand of aminopeptidase-Nreceptor (CD13). CD13 is a trans-membrane glycoprotein of 150 kDa highlyconserved in various species. It is expressed on normal cells as well asin myeloid tumor lines, in the angiogenic endothelium and is someepithelia. CD13 receptor is usually identified as “NGR” receptor, inthat its peptide ligands share the amino acidic “NGR” motif. We havealso surprisingly found that TNF coupled with a ligand of CD13 receptorand IFNγ act synergistically so that effective anti-tumor activity maybe seen upon co-administration at dosages which are below the effectivedoses individually. In addition, we have found that the anti-tumoractivity of a combination of the modified TNF and another anti-tumoragent, such as doxorubicin, is increased by administration of IFNγ.

STATEMENTS OF THE INVENTION

According to a first aspect, the invention provides a conjugationproduct of a cytokine selected from TNF and IFNγ and a ligand of CD13receptor.

According to another aspect of the present invention there is provided apharmaceutical composition comprising an effective amount of aconjugation product of TNF and a ligand of the CD13 receptor, and aneffective amount of IFNγ.

DETAILED DESCRIPTION OF THE INVENTION

Various preferred features and embodiments of the present invention willnow be described by way of non-limiting example.

Said ligand of CD13 receptor can be an antibody or a fragment thereofsuch as Fab, Fv, single-chain Fv, a peptide or a peptido-mimetic, namelya peptido-like molecule capable to bind the CD13 receptor, optionallycontaining modified, not naturally occurring amino acids.

CD13 is a trans-membrane glycoprotein of 150 kDa highly conserved invarious species. It is expressed on normal cells as well as in myeloidtumor lines, in the angiogenic endothelium and is some epithelia. CD13receptor is usually identified as “NGR” receptor. The ligands may benatural or synthetic. The term “ligand” also refers to a chemicallymodified ligand. The one or more binding domains of the ligand mayconsist of, for example, a natural ligand for the receptor, or afragment of a natural ligand which retains binding affinity for thereceptor. Synthetic ligands include the designer ligands. As usedherein, the term means “designer ligands” refers to agents which arelikely to bind to the receptor based on their three dimensional shapecompared to that of the receptor.

The ligand is preferably a straight or cyclic peptide comprising the NGRmotif, such as CNGRCVSGCAGRC (SEQ ID NO: 1), NGRAHA (SEQ ID NO: 2),GNGRG (SEQ ID NO: 3), cycloCVLNGRMEC (SEQ ID NO: 4) or cycloCNGRC (SEQID NO: 6), or, more preferably, the peptide CNGRC (SEQ ID NO: 5). Suchligands are described in WO98/10795 which is herein incorporated byreference. Methods of identifying ligands of CD13 receptor are disclosedin WO99/13329 which is herein incorporated by reference.

In one embodiment, the method of screening for an agent capable ofbinding to a CD13 receptor, the method comprising contacting the cellsurface molecule with an agent and determining if said agent binds tosaid cell surface molecule.

As used herein, the term “agent” includes, but is not limited to, acompound, such as a test compound, which may be obtainable from orproduced by any suitable source, whether natural or not. The agent maybe designed or obtained from a library of compounds which may comprisepeptides, as well as other compounds, such as small organic moleculesand particularly new lead compounds. By way of example, the agent may bea natural substance, a biological macromolecule, or an extract made frombiological materials such as bacteria, fungi, or animal (particularlymammalian) cells or tissues, an organic or an inorganic molecule, asynthetic test compound, a semi-synthetic test compound, a structural orfunctional mimetic, a peptide, a peptidomimetics, a derivatised testcompound, a peptide cleaved from a whole protein, or a peptidessynthesised synthetically (such as, by way of example, either using apeptide synthesizer) or by recombinant techniques or combinationsthereof, a recombinant test compound, a natural or a non-natural testcompound, a fusion protein or equivalent thereof and mutants,derivatives or combinations thereof.

The agent can be an amino acid sequence or a chemical derivativethereof. The substance may even be an organic compound or otherchemical.

As used herein the term “peptidomimetic” is used broadly to refer to apeptide-like molecule that has the binding activity of the CD13 ligand.

Alternatively, the ligand may be derived from heavy and light chainsequences from an immunoglobulin (Ig) variable region. Such a variableregion may be derived from a natural human antibody or an antibody fromanother species such as a rodent antibody. Alternatively the variableregion may be derived from an engineered antibody such as a humanisedantibody or from a phage display library from an immunised or anon-immunised animal or a mutagenised phage-display library. As a secondalternative, the variable region may be derived from a single-chainvariable fragment (scFv). The ligand may contain other sequences toachieve multimerisation or to act as spacers between the binding domainsor which result from the insertion of restriction sites in the genesencoding the ligand, including Ig hinge sequences or novel spacers andengineered linker sequences.

The ligand may comprise, in addition to one or more immunoglobulinvariable regions, all or part of an Ig heavy chain constant region andso may comprise a natural whole Ig, an engineered Ig, an engineeredIg-like molecule, a single-chain Ig or a single-chain Ig-like molecule.Alternatively, or in addition, the BP may contain one or more domainsfrom another protein such as a toxin.

As used herein, an “antibody” refers to a protein consisting of one ormore polypeptides substantially encoded by immunoglobulin genes orfragments of immunoglobulin genes. Antibodies may exist as intactimmunoglobulins or as a number of fragments, including thosewell-characterised fragments produced by digestion with variouspeptidases. While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate thatantibody fragments may be synthesised de novo either chemically or byutilising recombinant DNA methodology. Thus, the term antibody, as usedherein also includes antibody fragments either produced by themodification of whole antibodies or synthesised de novo usingrecombinant DNA methodologies. Antibody fragments encompassed by the useof the term “antibodies” include, but are not limited to, Fab, Fab′, F(ab′) 2, scFv, Fv, dsFv diabody, and Fd fragments.

If polyclonal antibodies are desired, a selected mammal (e.g., mouse,rabbit, goat, horse, etc.) is immunised with an immunogenic polypeptidebearing an epitope(s). Serum from the immunised animal is collected andtreated according to known procedures. If serum containing polyclonalantibodies to an epitope contains antibodies to other antigens, thepolyclonal antibodies can be purified by immunoaffinity chromatography.Techniques for producing and processing polyclonal antisera are known inthe art. In order that such antibodies may be made, the invention alsoprovides polypeptides of the invention or fragments thereof haptenisedto another polypeptide for use as immunogens in animals or humans.

Monoclonal antibodies directed against binding cell surface epitopes inthe polypeptides can also be readily produced by one skilled in the art.The general methodology for making monoclonal antibodies by hybridomasis well known. Immortal antibody-producing cell lines can be created bycell fusion, and also by other techniques such as direct transformationof B lymphocytes with oncogenic DNA, or transfection with Epstein-Barrvirus. Panels of monoclonal antibodies produced against epitopes can bescreened for various properties; i.e., for isotype and epitope affinity.

An alternative technique involves screening phage display librarieswhere, for example the phage express scFv fragments on the surface oftheir coat with a large variety of complementarity determining regions(CDRs). This technique is well known in the art.

For the purposes of this invention, the term “antibody”, unlessspecified to the contrary, includes fragments of whole antibodies whichretain their binding activity for a target antigen. As mentioned abovesuch fragments include Fv, F(ab′) and F(ab′)₂ fragments, as well assingle chain antibodies (scFv). Furthermore, the antibodies andfragments thereof may be humanised antibodies, for example as describedin EP-A-239400.

The term “peptide” as used herein includes polypeptides and proteins.The term “polypeptide” includes single-chain polypeptide molecules aswell as multiple-polypeptide complexes where individual constituentpolypeptides are linked by covalent or non-covalent means. The term“polypeptide” includes peptides of two or more amino acids in length,typically having more than 5, 10 or 20 amino acids.

It will be understood that polypeptide sequences for use in theinvention are not limited to the particular sequences or fragmentsthereof but also include homologous sequences obtained from any source,for example related viral/bacterial proteins, cellular homologues andsynthetic peptides, as well as variants or derivatives thereofPolypeptide sequences of the present invention also include polypeptidesencoded by polynucleotides of the present invention.

The terms “variant” or “derivative” in relation to the amino acidsequences of the present invention includes any substitution of,variation of, modification of, replacement of, deletion of or additionof one (or more) amino acids from or to the sequence providing theresultant amino acid sequence preferably has targeting activity,preferably having at least 25 to 50% of the activity as the polypeptidespresented in the sequence listings, more preferably at leastsubstantially the same activity.

Thus, sequences may be modified for use in the present invention.Typically, modifications are made that maintain the activity of thesequence. Thus, in one embodiment, amino acid substitutions may be made,for example from 1, 2 or 3 to 10, 20 or 30 substitutions provided thatthe modified sequence retains at least about 25 to 50% of, orsubstantially the same activity. However, in an alternative embodiment,modifications to the amino acid sequences of a polypeptide of theinvention may be made intentionally to reduce the biological activity ofthe polypeptide. For example truncated polypeptides that remain capableof binding to target molecule but lack functional effector domains maybe useful.

In general, preferably less than 20%, 10% or 5% of the amino acidresidues of a variant or derivative are altered as compared with thecorresponding region depicted in the sequence listings.

Amino acid substitutions may include the use of non-naturally occurringanalogues, for example to increase blood plasma half-life of atherapeutically administered polypeptide (see below for further detailson the production of peptide derivatives for use in therapy).

Conservative substitutions may be made, for example according to theTable below. Amino acids in the same block in the second column andpreferably in the same line in the third column may be substituted foreach other: ALIPHATIC Non-polar G A P I L V Polar - uncharged C S T M NQ Polar - charged D E K R AROMATIC H F W Y

Polypeptides of the invention also include fragments of the abovementioned polypeptides and variants thereof, including fragments of thesequences. Preferred fragments include those which include an epitope orbinding domain. Suitable fragments will be at least about 5, e.g. 10,12, 15 or 20 amino acids in length. They may also be less than 200, 100or 50 amino acids in length. Polypeptide fragments of the proteins andallelic and species variants thereof may contain one or more (e.g. 2, 3,5, or 10) substitutions, deletions or insertions, including conservedsubstitutions. Where substitutions, deletion and/or insertions have beenmade, for example by means of recombinant technology, preferably lessthan 20%, 10% or 5% of the amino acid residues depicted in the sequencelistings are altered.

Polypeptides and conjugates of the invention are typically made byrecombinant means, for example as described below. However they may alsobe made by synthetic means using techniques well known to skilledpersons such as solid phase synthesis. Various techniques for chemicalsynthesising peptides are reviewed by Borgia and Fields, 2000, TibTech18: 243-251 and described in detail in the references contained therein.

The peptide can be coupled directly to the cytokine or indirectlythrough a spacer, which can be a single amino acid, an amino acidsequence or an organic residue, such as6-aminocapryl-N-hydroxysuccinimide. The coupling procedures are known tothose skilled in the art and comprise genetic engineering or chemicalsynthesis techniques.

The peptide ligand preferably is linked to the cytokine N-terminus thusminimizing any interference in the binding of the modified cytokine toits receptor. Alternatively, the peptide can be linked to amino acidresidues which are amido- or carboxylic-bonds acceptors, naturallyoccurring on the molecule or artificially inserted with geneticengineering techniques. The modified cytokine is preferably prepared byuse of a cDNA comprising a 5′-contiguous sequence encoding the peptide.

According to a preferred embodiment, there is provided a conjugationproduct between TNF and the CNGRC (SEQ ID NO: 5) sequence. Morepreferably, the amino-terminal of TNF is linked to the CNGRC (SEQ ID NO:5) peptide through the spacer G (glycine).

The resulting product (NGR-TNF), proved to be more active than TNF onRMA-T lymphoma animal models. Furthermore, animals treated with NGR-TNFwere able to reject further tumorigenic doses of RMA-T or RMA cells. Theincrease in the antitumoral activity, as compared with normal TNF, couldbe observed in immunocompetent animals but not in immunodeficientanimals. This indicates that the increase in the antitumoral activity ofTNF conjugated with “NGR” peptides is due to an enhanced immune responserather than to a direct cytotoxic activity of the conjugate.

It has also been demonstrated that the in vivo immune effects induced byNGR-TNF are directly related to the CD13 receptor. It has, for example,been observed that antibody against the CD13 receptor as well as theGNGRC ligand compete with NGR-TNF in vivo, thus suggesting a mechanismof receptor targeting by NGR-TNF.

The therapeutic index of the TNF/CD13 ligand conjugates can be furtherimproved by using a mutant form of TNF capable of selectively binding toone of the two TNF receptors, p75TNFR and p55TNFR. Said TNF mutant canbe obtained by site-directed mutagenesis (5; 7).

The pharmacokinetic of the modified cytokines according to the inventioncan be improved by preparing polyethylene glycol derivatives, whichallow to extend the plasmatic half-life of the cytokines themselves.

A further embodiment of the invention is provided by bifunctionalderivatives in which the cytokines modified with the CD13 ligand areconjugated with antibodies, or their fragments, against tumoral antigensor other tumor angiogenic markers, e.g. αv integrins, metalloproteasesor the vascular growth factor, or antibodies or fragments thereofdirected against components of the extracellular matrix, such asanti-tenascin antibodies or anti-fibronectin EDB domain. The preparationof a fusion product between TNF and the hinge region of a mAb againstthe tumor-associated TAG72 antigen expressed by gastric and ovarianadenocarcinoma has recently been reported (6).

A further embodiment of the invention is provided by the tumoralpre-targeting with the biotin/avidin system. According to this approach,a ternary complex is obtained on the tumoral antigenic site, atdifferent stages, which is formed by 1) biotinylated mAb, 2) avidin (orstreptavidin) and 3) bivalent cytokine modified with the CD13 ligand andbiotin. A number of papers proved that the pre-targeting approach,compared with conventional targeting with immunoconjugates, can actuallyincrease the ratio of active molecule homed at the target to free activemolecule, thus reducing the treatment toxicity (11, 10, 9, 8). Thisapproach produced favorable results with biotinylated TNF, which wascapable of inducing cytotoxicity in vitro and decreasing the tumor cellsgrowth under conditions in which normal TNF was inactive (14, 26). Thepre-targeting approach can also be carried out with a two-phaseprocedure by using a bispecific antibody which at the same time bindsthe tumoral antigen and the modified cytokine. The use of a bispecificantibody directed against a carcinoembryonic antigen and TNF hasrecently been described as a means for TNF tumoral pre-targeting (31).

According to a further embodiment, the invention comprises a TNFmolecule conjugated to both a CD13 ligand and an antibody, or a fragmentthereof (directly or indirectly via a biotin-avidin bridge), ondifferent TNF subunits, where the antibody or its fragments are directedagainst an antigen expressed on tumor cells or other components of thetumor stroma, e.g. tenascin and fibronectin EDB domain. This results ina further improvement of the tumor homing properties of the modifiedcytokine and in the slow release of the latter in the tumormicroenvironment through trimer-monomer-trimer transitions. As shown inprevious works, in fact, the modified subunits of TNF conjugates candissociate from the targeting complexes and reassociate so as to formunmodified trimeric TNF molecules, which then diffuse in the tumormicroenvironment. The release of bioactive TNF has been shown to occurwithin 24-48 hours after targeting (21).

Peptides of the present invention may be administered therapeutically topatients. It is preferred to use peptides that do not consisting solelyof naturally-occurring amino acids but which have been modified, forexample to reduce immunogenicity, to increase circulatory half-life inthe body of the patient, to enhance bioavailability and/or to enhanceefficacy and/or specificity.

A number of approaches have been used to modify peptides for therapeuticapplication. One approach is to link the peptides or proteins to avariety of polymers, such as polyethylene glycol (PEG) and polypropyleneglycol (PPG)—see for example U.S. Pat. Nos. 5,091,176, 5,214,131 andU.S. Pat. No. 5,264,209.

Replacement of naturally-occurring amino acids with a variety of uncodedor modified amino acids such as D-amino acids and N-methyl amino acidsmay also be used to modify peptides

Another approach is to use bifunctional crosslinkers, such asN-succinimidyl 3-(2 pyridyldithio)propionate, succinimidyl 6-[3-(2pyridyldithio)propionamido]hexanoate, and sulfosuccinimidyl 6-[3-(2pyridyldithio)propionamido]hexanoate (see U.S. Pat. No. 5,580,853).

It may be desirable to use derivatives of the peptides of the inventionwhich are conformationally constrained. Conformational constraint refersto the stability and preferred conformation of the three-dimensionalshape assumed by a peptide. Conformational constraints include localconstraints, involving restricting the conformational mobility of asingle residue in a peptide; regional constraints, involving restrictingthe conformational mobility of a group of residues, which residues mayform some secondary structural unit; and global constraints, involvingthe entire peptide structure.

The active conformation of the peptide may be stabilised by a covalentmodification, such as cyclization or by incorporation of gamma-lactam orother types of bridges. For example, side chains can be cyclized to thebackbone so as create a L-gamma-lactam moiety on each side of theinteraction site. See, generally, Hruby et al., “Applications ofSynthetic Peptides,” in Synthetic Peptides: A User's Guide: 259-345 (W.H. Freeman & Co. 1992). Cyclization also can be achieved, for example,by formation of cysteine bridges, coupling of amino and carboxy terminalgroups of respective terminal amino acids, or coupling of the aminogroup of a Lys residue or a related homolog with a carboxy group of Asp,Glu or a related homolog. Coupling of the alpha-amino group of apolypeptide with the epsilon-amino group of a lysine residue, usingiodoacetic anhydride, can be also undertaken. See Wood and Wetzel, 1992,Int'l J. Peptide Protein Res. 39: 533-39.

Another approach described in U.S. Pat. No. 5,891,418 is to include ametal-ion complexing backbone in the peptide structure. Typically, thepreferred metal-peptide backbone is based on the requisite number ofparticular coordinating groups required by the coordination sphere of agiven complexing metal ion. In general, most of the metal ions that mayprove useful have a coordination number of four to six. The nature ofthe coordinating groups in the peptide chain includes nitrogen atomswith amine, amide, imidazole, or guanidino functionalities; sulfur atomsof thiols or disulfides; and oxygen atoms of hydroxy, phenolic,carbonyl, or carboxyl functionalities. In addition, the peptide chain orindividual amino acids can be chemically altered to include acoordinating group, such as for example oxime, hydrazino, sulfhydryl,phosphate, cyano, pyridino, piperidino, or morpholino. The peptideconstruct can be either linear or cyclic, however a linear construct istypically preferred. One example of a small linear peptide is a sequenceof four glycine residues which has four nitrogens (an N₄ complexationsystem) in the back bone that can complex to a metal ion with acoordination number of four.

A further technique for improving the properties of therapeutic peptidesis to use non-peptide peptidomimetics. A wide variety of usefultechniques may be used to elucidating the precise structure of apeptide. These techniques include amino acid sequencing, x-raycrystallography, mass spectroscopy, nuclear magnetic resonancespectroscopy, computer-assisted molecular modelling, peptide mapping,and combinations thereof. Structural analysis of a peptide generallyprovides a large body of data which comprise the amino acid sequence ofthe peptide as well as the three-dimensional positioning of its atomiccomponents. From this information, non-peptide peptidomimetics may bedesigned that have the required chemical functionalities for therapeuticactivity but are more stable, for example less susceptible to biologicaldegradation. An example of this approach is provided in U.S. Pat. No.5,811,512.

Techniques for chemically synthesising therapeutic peptides of theinvention are described in the above references and also reviewed byBorgia and Fields, 2000, TibTech 18: 243-251 and described in detail inthe references contained therein.

For use in therapy, the modified cytokines of the invention will besuitably formulated in pharmaceutical preparations for the oral orparenteral administration. Formulations for the parenteraladministration are preferred, and they comprise injectable solutions orsuspensions and liquids for infusions. For the preparation of theparenteral forms, an effective amount of the active ingredient will bedissolved or suspended in a sterile carrier, optionally addingexcipients such as solubilizers, isotonicity agents, preservatives,stabilizers, emulsifiers or dispersing agents, and it will besubsequently distributed in sealed vials or ampoules.

In more detail, conjugates of the invention, including polypeptides andpolynucleotides, may preferably be combined with various components toproduce compositions of the invention. Preferably the compositions arecombined with a pharmaceutically acceptable carrier, diluent orexcipient to produce a pharmaceutical composition (which may be forhuman or animal use). Suitable carriers and diluents include isotonicsaline solutions, for example phosphate-buffered saline. Details ofexcipients may be found in The Handbook of Pharmaceutical Excipients,2nd Edn, Eds Wade & Weller, American Pharmaceutical Association. Thecomposition of the invention may be administered by direct injection.The composition may be formulated for parenteral, intramuscular,intravenous, subcutaneous, intraocular, oral or transdermaladministration.

The composition may be formulated such that administration daily, weeklyor monthly will provide the desired daily dosage. It will be appreciatedthat the composition may be conveniently formulated for administratedless frequently, such as every 2, 4, 6, 8, 10 or 12 hours.

Polynucleotides/vectors encoding polypeptide components may beadministered directly as a naked nucleic acid construct, preferablyfurther comprising flanking sequences homologous to the host cellgenome.

Uptake of naked nucleic acid constructs by mammalian cells is enhancedby several known transfection techniques for example those including theuse of transfection agents. Example of these agents include cationicagents (for example calcium phosphate and DEAE-dextran) and lipofectants(for example lipofectam™ and transfectam™). Typically, nucleic acidconstructs are mixed with the transfection agent to produce acomposition.

Preferably the polynucleotide or vector of the invention is combinedwith a pharmaceutically acceptable carrier or diluent to produce apharmaceutical composition. Suitable carriers and diluents includeisotonic saline solutions, for example phosphate-buffered saline. Thecomposition may be formulated for parenteral, intramuscular,intravenous, subcutaneous, intraocular or transdermal administration.

The routes of administration and dosage regimens described are intendedonly as a guide since a skilled practitioner will be able to determinereadily the optimum route of administration and dosage regimens for anyparticular patient and condition.

The preparation of cytokines in form of liposomes can improve thebiological activity thereof. It has, in fact, been observed thatacylation of the TNF amino groups induces an increase in itshydrophobicity without loss of biological activity in vitro.Furthermore, it has been reported that TNF bound to lipids hasunaffected cytotoxicity in vitro, immunomodulating effects and reducedtoxicity in vivo (12, 13).

The maximum tolerated dose of bolus TNF in humans is 218-410 μg/m² (32)about 10-fold lower than the effective dose in animals. Based on datafrom murine models it is believed that an at least 10 times higher doseis necessary to achieve anti-tumor effects in humans (15). In the firstclinical study on hyperthermic isolated-limb perfusion, high responserates were obtained with the unique dose of 4 mg of TNF in combinationwith melphalan and interferon γ (16). Other works showed that interferony can be omitted and that even lower doses of TNF can be sufficient toinduce a therapeutic response (17, 18). As the two cytokines exertsynergistic effects on endothelial cells, their combined, selectivetargeting thereon is likely to result in stronger anti-tumor activitythus allowing to overcome the problems of systemic toxicity usuallyencountered in cancer therapy with the same cytokines used incombination. Furthermore, it is known that TNF can decrease the barrierfunction of the endothelial lining vessels, thus increasing theirpermeability to macromolecules. Taking advantage of the lower toxicityof treatment with the modified TNF molecules according to the invention,and of their tumor vessels homing properties, an alternative applicationis their use to increase the permeability of tumor vessels to othercompounds, either for therapeutic or diagnostic purposes. For instancethe modified TNF can be used to increase the tumor uptake ofradiolabelled antibodies or hormones (tumor-imaging compounds) inradioimmunoscintigraphy or radioimmunotherapy of tumors. Alternatively,the uptake of chemotherapeutic drugs, immunotoxins, liposomes carryingdrugs or genes, or other anticancer drugs could also be increased, sothat their antitumor effects are enhanced.

Accordingly, the cytokines of the invention can be used in combined,separated or sequential preparations, also with other diagnostic ortherapeutic substances, in the treatment or in the diagnosis of cancer.

Another aspect of the present invention relates to the use of acombination of the modified TNF, and IFNγ. This combination can be usedin combined, separated or sequential preparations. Advantageously thecombination is also with other diagnostic or therapeutic substances, inthe treatment or in the diagnosis of cancer, such as doxorubicin andmephalan. Thus the present invention provides a pharmaceuticalcomposition comprising a combination of the modified TNF and IFNγ, andoptionally another tumor-diagnostic or anti-tumor therapeutic substance.Again, this combination can be used in combined, separated or sequentialpreparations.

In our patent application number GB 02098960, we found targeted deliveryof picogram doses of cytokines enhances the penetration ofchemotherapeutic drugs, providing a novel and surprising strategy forincreasing the therapeutic index of chemotherapeutic drugs. Patentapplication number GB 02098960 is hereby incorporated by reference inits entirety. In more detail, we have found that delivery of very lowdoses of cytokines to tumors and the tumor-associated environmentincluding tumor vasculature represents a new approach to avoidingnegative feedback mechanisms and to preserve its ability to alterdrug-penetration barriers.

The composition of the present invention may be formulated forparenteral, intramuscular, intravenous, subcutaneous, intraocular, oralor transdermal administration. In one embodiment of this aspect of thepresent invention, a conjugate of the present invention may beadministered at a dose of from in the range of 0.5 to 500 ng/kg,preferably in the range of 1 to 50 ng/kg, more preferably in the rangeof 5 to 15 ng/kg.

In an alternative embodiment of this aspect of the invention there isprovided a pharmaceutical composition comprising a conjugate of thepresent invention in combination with IFNγ, wherein the conjugate ispresent in an amount such that the conjugate or a metabolite thereof isprovided to the blood plasma of the subject to be treated in an amountof no greater than about 35,000 ng/day, preferably about 3,500 ng/day,more preferably about 1,000ng/day.

The above dosage relate to a dosage for a 70 kg subject. A personskilled in the art would readily be able to modify the recited dosagefor a subject having as mass other than 70 kg.

The routes of administration and dosage regimens described are intendedonly as a guide since a skilled practitioner will be able to determinereadily the optimum route of administration and dosage regimens for anyparticular patient and condition.

Another aspect of the invention regards the cDNA encoding for theconjugated cytokines herein disclosed, which can be prepared from thecytokines cDNA by addition of a 5′- or 3′-contiguous DNA sequenceencoding for the CD13 ligand, preferably for the homing peptidesdescribed above. The combined cDNA can be used as such or afterinsertion in vectors for gene therapy. The preparation and therapeuticapplications of suitable vectors is disclosed in (19), which is herebyincorporated by reference.

Polynucleotides for use in the invention comprise nucleic acid sequencesencoding the polypeptide conjugate of the invention. It will beunderstood by a skilled person that numerous different polynucleotidescan encode the same polypeptide as a result of the degeneracy of thegenetic code. In addition, it is to be understood that skilled personsmay, using routine techniques, make nucleotide substitutions that do notaffect the polypeptide sequence encoded by the polynucleotides of theinvention to reflect the codon usage of any particular host organism inwhich the polypeptides of the invention are to be expressed.

Polynucleotides of the invention may comprise DNA or RNA. They may besingle-stranded or double-stranded. They may also be polynucleotideswhich include within them synthetic or modified nucleotides. A number ofdifferent types of modification to oligonucleotides are known in theart. These include methylphosphonate and phosphorothioate backbones,addition of acridine or polylysine chains at the 3′ and/or 5′ ends ofthe molecule. For the purposes of the present invention, it is to beunderstood that the polynucleotides described herein may be modified byany method available in the art. Such modifications may be carried outin order to enhance the in vivo activity or life span of polynucleotidesof the invention.

Polynucleotides of the invention can be incorporated into a recombinantreplicable vector. The vector may be used to replicate the nucleic acidin a compatible host cell. Thus in a further embodiment, the inventionprovides a method of making polynucleotides of the invention byintroducing a polynucleotide of the invention into a replicable vector,introducing the vector into a compatible host cell, and growing the hostcell under conditions which bring about replication of the vector. Thevector may be recovered from the host cell. Suitable host cells includebacteria such as E. Coli, yeast, mammalian cell lines and othereukaryotic cell lines, for example insect Sf9 cells.

Preferably, a polynucleotide of the invention in a vector is operablylinked to a control sequence that is capable of providing for theexpression of the coding sequence by the host cell, i.e. the vector isan expression vector. The term “operably linked” means that thecomponents described are in a relationship permitting them to functionin their intended manner. A regulatory sequence “operably linked” to acoding sequence is ligated in such a way that expression of the codingsequence is achieved under condition compatible with the controlsequences.

The control sequences may be modified, for example by the addition offurther transcriptional regulatory elements to make the level oftranscription directed by the control sequences more responsive totranscriptional modulators.

Vectors of the invention may be transformed or transfected into asuitable host cell as described below to provide for expression of aprotein of the invention. This process may comprise culturing a hostcell transformed with an expression vector as described above underconditions to provide for expression by the vector of a coding sequenceencoding the protein, and optionally recovering the expressed protein.

The vectors may be for example, plasmid or virus vectors provided withan origin of replication, optionally a promoter for the expression ofthe said polynucleotide and optionally a regulator of the promoter. Thevectors may contain one or more selectable marker genes, for example anampicillin resistance gene in the case of a bacterial plasmid or aneomycin resistance gene for a mammalian vector. Vectors may be used,for example, to transfect or transform a host cell.

Control sequences operably linked to sequences encoding the protein ofthe invention include promoters/enhancers and other expressionregulation signals. These control sequences may be selected to becompatible with the host cell for which the expression vector isdesigned to be used in. The term “promoter” is well-known in the art andencompasses nucleic acid regions ranging in size and complexity fromminimal promoters to promoters including upstream elements andenhancers.

The promoter is typically selected from promoters which are functionalin mammalian cells, although prokaryotic promoters and promotersfunctional in other eukaryotic cells may be used. The promoter istypically derived from promoter sequences of viral or eukaryotic genes.For example, it may be a promoter derived from the genome of a cell inwhich expression is to occur. With respect to eukaryotic promoters, theymay be promoters that function in a ubiquitous manner (such as promotersof a-actin, b-actin, tubulin) or, alternatively, a tissue-specificmanner (such as promoters of the genes for pyruvate kinase).Tissue-specific promoters specific for certain cells may also be used.They may also be promoters that respond to specific stimuli, for examplepromoters that bind steroid hormone receptors. Viral promoters may alsobe used, for example the Moloney murine leukaemia virus long terminalrepeat (MMLV LTR) promoter, the rous sarcoma virus (RSV) LTR promoter orthe human cytomegalovirus (CMV) IE promoter.

It may also be advantageous for the promoters to be inducible so thatthe levels of expression of the heterologous gene can be regulatedduring the life-time of the cell. Inducible means that the levels ofexpression obtained using the promoter can be regulated.

In addition, any of these promoters may be modified by the addition offurther regulatory sequences, for example enhancer sequences. Chimericpromoters may also be used comprising sequence elements from two or moredifferent promoters described above.

Vectors and polynucleotides of the invention may be introduced into hostcells for the purpose of replicating the vectors/polynucleotides and/orexpressing the proteins of the invention encoded by the polynucleotidesof the invention. Although the proteins of the invention may be producedusing prokaryotic cells as host cells, it is preferred to use eukaryoticcells, for example yeast, insect or mammalian cells, in particularmammalian cells.

Vectors/polynucleotides of the invention may introduced into suitablehost cells using a variety of techniques known in the art, such astransfection, transformation and electroporation. Wherevectors/polynucleotides of the invention are to be administered toanimals, several techniques are known in the art, for example infectionwith recombinant viral vectors such as retroviruses, herpes simplexviruses and adenoviruses, direct injection of nucleic acids andbiolistic transformation.

Host cells comprising polynucleotides of the invention may be used toexpress conjugates of the invention. Host cells may be cultured undersuitable conditions which allow expression of the polypeptides andconjugates of the invention. Expression of the products of the inventionmay be constitutive such that they are continually produced, orinducible, requiring a stimulus to initiate expression. In the case ofinducible expression, protein production can be initiated when requiredby, for example, addition of an inducer substance to the culture medium,for example dexamethasone or IPTG.

Conjugates of the invention can be extracted from host cells by avariety of techniques known in the art, including enzymatic, chemicaland/or osmotic lysis and physical disruption.

DESCRIPTION OF THE FIGURES

FIG. 1: Effect of TNF and NGR-TNF on the growth of RMA-T lymphomas (aand b) and on the animal weight (c and d).

5 Animals/group were treated with a single dose of TNF or NGR-TNF(i.p.), 10 days after tumor implantation. Tumor area values at day 14 asa function of the dose (b) and the loss of weight after treatment (meanof days 11 and 12) (d), were interpolated from logarithmic curves. Theanti-tumor effects induced by 1 μg or 9 μg of NGR-TNF at day 14 weregreater than those induced by comparable amounts of TNF (P=0.024 andP=0.032, respectively), while the loss of weight after these treatmentswas similar. The arrows indicate extrapolated doses of TNF and NGR-TNFthat induce comparable effects.

FIG. 2: Effect of mAb R3-63 and CNGRC on the anti-tumor activity ofNGR-TNF (a) and TNF (b).

MAb R3-63 or CNGRC (SEQ ID NO: 5) were mixed with NGR-TNF or TNF andadministered to RMA-T tumor bearing animals, 12 days after tumorimplantation (n=5 animals/group). In a separate experiment (c) TNF andNGR-TNF were coadministered with CNGRC (SEQ ID NO: 5) or CARAC SEQ IDNO: 19) (a control peptide) to animals bearing 11-day old tumors (n=5).The anti-tumor effect of 1 μg of NGR-TNF was stronger than that of 9 μgof TNF (P=0.009, t-test at day 20) and was significantly inhibited byCNGRC (SEQ ID NO: 5) (P=0.035) and by mAb R3-63 (P=0.011).

FIG. 3: Effect of limited tryptic digestion of NGR-TNF and TNF on theirmass (a) and anti-tumor activity (b).

Trypsin-agarose was prepared by coupling 1 mg of trypsin to 1 ml ofActivated CH Sepharose (Pharmacia-Upjohn), according to themanufacturer's instructions. NGR-TNF and TNF (170 μg each in 300 μL of0.15 M sodium chloride, 0.05 M sodium phosphate, pH 7.3) were mixed with15 μl of resin suspension (1:4) or buffer alone and rotated end-over-endat 37° C. for the indicated time. The four products were filteredthrough a 0.22 μm Spin-X device (Costar, Cambridge, Mass.) and stored at−20° C. until use. (a) Electrospray mass spectrometry analysis. Themolecular mass values and the corresponding products (N-terminalsequences) are indicated on each peak. The arrows on the sequencesindicate the site of cleavage. (b) Effect of 1 or 3 μg of NGR-TNF andTNF, incubated without (upper panels) or with (lower panels) trypsin, onthe growth of RMA-T tumors and animal weight (mean±SE of groups treatedwith 1 and 3 μg doses). Animals were treated 13 days after tumorimplantation (n=5 animals/group).

FIG. 4: SDS-PAGE and anti-tumor activity of human NGR-TNF before andafter denaturation/refolding.

SDS-PAGE under non reducing conditions (A) of human TNF (a), NGR-TNFbefore (b) and after (c) the denaturation/refolding process described inExample V.

Effect of TNF and non-refolded NGR-TNF on the growth of RMA-T lymphomas(B) and on body weight (C). Effect of human TNF (D) and refolded NGR-TNF(consisting of >95% trimers with intra-chain disulfides) (E) on thetumor growth. Animals (15 or 5 mice/group as indicated in each panel)were treated with one i.p. dose of TNF or NGR-TNF, 10 days after tumorimplantation.

FIG. 5: Effect of a neutralising anti-mIFNγ antibody (AN18) on theantitumor activity of NGR-mTNF and doxorubicin on B16F1 tumors in C57BL6mice.

FIG. 6: Effect of NGR-TNF and doxorubicin in IFN knock out mice.

FIG. 7: Effect of NGR-mTNF, mIFNγ and doxorubicin (alon or incombinantion) on B16F1 tumors in nude mice.

FIG. 8: Effect of NGR-TNF and doxorubicin in nude mice

FIG. 9: Effect of NGR-mTNF, mIFNγ and doxorubicin on B16F1 tumors inimmunocompetent mice (C57BL6).

FIG. 10: IFN increase the penetration of doxorubicin in tumours whencombined with NGR-TNF in IFN knock out animals.

The following examples further illustrate the invention.

EXAMPLE I

Preparation of Murine TNF and NGR-TNF

Murine recombinant TNF and Cys-Asn-Gly-Arg-Cys-Gly-TNF (NGR-TNF) (SEQ IDNO: 18) were produced by cytoplasmic cDNA expression in E. coli. ThecDNA coding for murine Met-TNF₁₋₅₆ (20) was prepared by reversetranscriptase-polymerase chain reaction (RT-PCR) on mRNA isolated fromlipopolysaccharide-stimulated murine RAW-264.7 monocyte-macrophagecells, using 5′-CTGGATCCTCACAGAGCAATGACTCCAAAG-3′ (SEQ ID NO:7) and5′-TGCCTCACATATGCTCAGATCATCTTCTC-3′, (SEQ ID NO:8) as 3′ and 5′ primers.

The amplified fragment was digested with Nde I and Bam HI (New EnglandBiolabs, Beverley, Mass.) and cloned in pET-11b (Novagen, Madison,Wis.), previously digested with the same enzymes (pTNF).

The cDNA coding for Cys-Asn-Gly-Arg-Cys-Gly-TNF₁₋₅₆ (SEQ ID NO: 18) wasamplified by PCR on pTNF, using5′-GCAGATCATATGTGCAACGGCCGTTGCGGCCTCAGATCATCTTCTC-3′ (SEQ ID NO: 9) as5′ primer, and the above 3′ primer. The amplified fragment was digestedand cloned in pET-11b as described above and used to transform BL21(DE3)E. coli cells (Novagen). The expression of TNF and NGR-TNF was inducedwith isopropyl-β-D-thiogalactoside, according to the pET11bmanufacturer's instruction. Soluble TNF and NGR-TNF were recovered fromtwo-liter cultures by bacterial sonication in 2 mMethylenediaminetetracetic acid, 20 mM Tris-HCl, pH 8.0, followed bycentrifugation (15000×g, 20 min, 4° C.). Both extracts were mixed withammonium sulfate (25% of saturation), left for 1 h at 4° C., and furthercentrifuged, as above. The ammonium sulfate in the supernatants was thenbrought to 65% of saturation, left at 4° C. for 24 h and furthercentrifuged. Each pellet was dissolved in 200 ml of 1 M ammoniumsulfate, 50 mM Tris-HCl, pH 8.0, and purified by hydrophobic interactionchromatography on Phenyl-Sepharose 6 Fast Flow (Pharmacia-Upjohn)(gradient elution, buffer A: 50 mM sodium phosphate, pH 8.0, containing1 M ammonium sulfate; buffer B: 20% glycerol, 5% methanol, 50 mM sodiumphosphate, pH 8.0). Fractions containing TNF immunoreactive material (bywestern blotting) were pooled, dialyzed against 2 mMethylenediaminetetracetic acid, 20 mM Tris-HCl, pH 8.0 and furtherpurified by ion exchange chromatography on DEAE-Sepharose Fast Flow(Pharmacia-Upjohn) (gradient elution, buffer A: 20 mM Tris-HCl, pH 8.0;buffer B: 1 M sodium chloride, 20 mM Tris-HCl, pH 8.0). Fractionscontaining TNF-immunoreactivity were pooled and purified by gelfiltration chromatography on Sephacryl-S-300 HR (Pharmacia-Upjohn),pre-equilibrated and eluted with 150 mM sodium chloride, 50 mM sodiumphosphate buffer, pH 7.3 (PBS). Fractions corresponding to 40000-50000Mr products were pooled, aliquoted and stored frozen at −20° C. Allsolutions employed in the chromatographic steps were prepared withsterile and endotoxin-free water (Salf, Bergamo, Italy). The finalyields were 45 mg of TNF and 34.5 mg NGR-TNF.

The molecular weight of purified TNF and NGR-TNF was measured byelectrospray mass spectrometry. The protein content was measured using acommercial protein assay kit (Pierce, Rockford, Ill.). Endotoxin contentof NGR-TNF and TNF was 0.75 units/μg and 1.38 units/μg, respectively, asmeasured by the quantitative chromogenic Lymulus Amoebocyte Lysate (LAL)test (BioWhittaker).

Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) andwestern blot analysis were carried out using 12.5 or 15% polyacrylamidegels, by standard procedures.

A small amount of TNF and NGR-TNF was further purified by affinitychromatography on soluble p55-TNF receptor (sTNF-R1)-Sepharose asfollows: 5 mg of recombinant sTNF-R1 were prepared as described (22) andcoupled to 2 ml of Activated-CH-Sepharose (Pharmacia), according to themanufacturer's instruction. Two separate columns (one ml each), werewashed extensively with sterile and endotoxin-free solutions, loadedwith purified TNF or NGR-TNF in PBS and desorbed by gradient elution (1h, buffer A: PBS; buffer B: 0.5 M sodium chloride, 0.2 M glycine-HCl).The TNF-antigen containing fractions were neutralized and dialyzedagainst sterile physiological solution. Endotoxin-free human serumalbumin was added before dialysis (0.5 mg/ml) to prevent proteinadsorption on membranes. The TNF content in each fraction was measuredby ELISA and cytolytic assay.

Non reducing SDS-PAGE of TNF showed a single band of 17-18 kDa, asexpected for monomeric TNF (not shown). At variance, non reducingSDS-PAGE and western blot analysis of NGR-TNF showed differentimmunoreactive forms of 18, 36 and 50 kDa likely corresponding tomonomers, dimers and trimers. Under reducing conditions most of the 50and 36 kDa bands were converted into the 18 kDa form, pointing to thepresence of NGR-TNF molecules with interchain disulfide bridges. The 18kDa band accounted to about ⅔ of the total material, whereas the 36 kDaaccounted for most of the remaining part. These electrophoretic patternssuggest that NGR-TNF was a mixture of trimers made up by three monomericsubunits with correct intra-chain disulfides (at least 50%) and theremaining part mostly by trimers with one or more interchain disulfides.The 36 kDa band still observed by reducing SDS-PAGE suggests thatNGR-TNF contained also an irreversible denatured dimer (about 10% oftotal).

The molecular mass of TNF and NGR-TNF monomers were 17386.1±2.0 Da and17843.7±2.5 Da, respectively, by electrospray mass spectrometry. Thesevalues correspond very well to the mass expected for Met-TNF₁₋₁₅₆(17386.7 Da) and for CNGRCG-TNF₁₋₁₅₆ (17844.2 Da).

EXAMPLE II

In Vitro Cytotoxic Activity of Murine TNF and NGR-TNF

The bioactivity of TNF and NGR-TNF was estimated by standard cytolyticassay based on L-M mouse fibroblasts (ATCC CCL1.2) as described (23).The cytolytic activity of TNF and NGR-TNF on RMA-T cells was tested inthe presence of 30 ng/ml actinomycin D. Each sample was analyzed induplicate, at three different dilutions. The results are expressed asmean±SD of two-three independent assays.

The in vitro cytotoxic activity of TNF and NGR-TNF was (1.2±0.14)×10⁸units/mg and (1.8±0.7)×10⁸ units/mg, respectively, by standard cytolyticassay with L-M cells. These results indicate that the CNGRCG (SEQ ID NO:18) moieties in the NGR-TNF molecule does not prevent folding,oligomerization and binding to TNF receptors.

In a previous study we showed that RMA-T cells can be killed by TNF inthe presence of 30 ng/ml actinomycin D, whereas in the absence oftranscription inhibitors these cells are resistant to TNF, even afterseveral days of incubation. The in vitro cytotoxic activity of NGR-TNFon RMA-T cells in the presence of actinomycin D was (1.4±0.8)×10⁸units/mg, as measured using TNF ((1.2±0.14)×10⁸ units/mg) as a standard.Thus, the cytotoxic activities of NGR-TNF and TNF were similar both onL-M and RMA-T cells.

EXAMPLE III

Characterization of the Therapeutic and Toxic Activity of Murine TNF andNGR-TNF

The Rauscher virus-induced RMA lymphoma of C57BL/6 origin, weremaintained in vitro in RPMI 1640, 5% foetal bovine serum (FBS), 100 U/mlpenicillin, 100 μg/ml streptomycin, 0.25 μg/ml amphotericin B, 2 mMglutamine and 50 μM 2-mercaptoethanol. RMA-T was derived from the RMAcell line by transfection with a construct encoding the Thy 1.1 alleleand cultured as described (14).

B16F1 melanoma cells were cultured in RPMI 1640, 5% FBS, 100 U/mlpenicillin, 100 μg/ml streptomycin, 0.25 μg/ml amphotericin B, 2 mMglutamine, 1% MEM non essential amino acid (BioWhittaker Europe,Verviers, Belgium).

In vivo studies on animal models were approved by the Ethical Committeeof the San Raffaele H Scientific Institute and performed according tothe prescribed guidelines. C57BL/6 (Charles River Laboratories, Calco,Italy) (16-18 g) were challenged with 5×10⁴ RMA-T or B16F1 living cells,respectively, s.c. in the left flank. Ten-twelve days after tumorimplantation, mice were treated, i.p., with 250 μl TNF or NGR-TNFsolutions, diluted with endotoxin-free 0.9% sodium chloride. Preliminaryexperiments showed that the anti-tumor activity was not changed by theaddition of human serum albumin to TNF and NGR-TNF solutions, as acarrier. Each experiment was carried out with 5 mice per group. Thetumor growth was monitored daily by measuring the tumor size withcalipers. The tumor area was estimated by calculating r₁×r₂, whereastumor volume was estimated by calculating r₁×r₂×r₃× 4/3, where r₁ and r₂are the longitudinal and lateral radii, and r₃ is the thickness oftumors protruding from the surface of normal skin. Animals were killedbefore the tumor reached 1.0-1.3 cm diameter. Tumor sizes are shown asmean±SE (5-10 animals per group as indicated in the figure legends) andcompared by t-test.

The anti-tumor activity and toxicity of NGR-TNF were compared to thoseof TNF using the RMA-T lymphoma and the B16F1 melanoma models in C57BL6mice. Since the RMA-T model has been previously characterized and usedto study the anti-tumor activity of TNF with different targetingprotocols (26) we decided to use this model also in this study.

Murine TNF administered to animals bearing established s.c. RMA-Ttumors, causes 24 h later a reduction in the tumor mass and haemorragicnecrosis in the central part of the tumor, followed by a significantgrowth delay for few days (26). A single treatment with TNF does notinduce complete regression of this tumor, even at doses close to theLD50, as living cells remaining around the necrotic area restart to growfew days after treatment.

In a first set of experiments we investigated the effect of variousdoses (i.p.) of TNF or NGR-TNF on animal survival. To avoid excessivesuffering, the animals were killed when the tumor diameter was greaterthan 1-1.3 cm. The lethality of TNF and NGR-TNF, 3 days after treatment,was similar (LD50, 60 μg and 45 μg, respectively) whereas theiranti-tumor activity was markedly different (Table 1). TABLE 1 Survivalof mice with RMA-T lymphoma treated with murine TNF or NGR-TNF Survival(%)^(a)) after treatment Animals Dose Day Day Day Day Day Day 38 Day 62Day Treatment (n) (□g) 3 7 14 21 30 (2^(nd) ch)^(b)) (3° ch.)^(b)) 92None 18 0 100 0 TNF 4 1 100 20 0 9 3 100 55 0 9 9 100 55 22 11 0 14 27100 57 14 7 0 9 54 55 55 0 9 108 0 NGR-TNF 10 1 100 70 10 10 10 0 10 3100 80 20 20 20 0 9 9 100 88 55 22 11 11 11 13 27 100 85 30 23 15 15 1511 9 54 33 33 0 15 9 108 0^(a))Animals with tumor were treated with TNF or NGR-TNF (i.p.) 10 daysafter tumor implant. Animals were killed when the tumor diameterexceeded 1-1.3 cm.^(b))Surviving animals were re-challenged with 50,000 RMA-T cells(second challenge) or 50,000 RMA (third) at the indicated time.Tumorigenicity of injected cells was monitored at each time with the 5normal animals. All control animals developed a tumor within 10 days.(data not shown).

For instance, 1 or 3 μg of NGR-TNF delayed tumor growth more efficientlythen 27 μg of TNF, indicating that NGR-TNF was at least one order ofmagnitude more active. Interestingly, some animals were cured with dosesof NGR-TNF lower than the LD50, whereas no animals at all were curedwith TNF. Cured animals rejected further challenges with tumorigenicdoses of either RMA-T or wild-type RMA cells, suggesting that a singletreatment with NGR-TNF was able to induce protective immunity. It isnoteworthy that increasing the dose of TNF or NGR-TNF above 9-27 μgmarkedly increased the toxicity and poorly or not the therapeuticactivity.

The loss of weight consequent to TNF treatment is a well known sign ofsystemic toxicity (26 ). Thus, to further compare the efficacy/toxicityratio of TNF and NGR-TNF we monitored the tumor growth and the animalweight after treatment. The effect of 1 μg of NGR-TNF on the tumorgrowth was similar or higher than that of 9 μg of TNF (FIG. 1 a), whilethe loss of weight one-two days after treatment was comparable to thatof 1 μg of TNF (FIG. 1 c). When we interpolated the data with alogarithmic curve in a dose-response plot we found that the therapeuticeffect of 9 μg of TNF at day 14 can be obtained with as little as 0.6 μgof NGR-TNF (FIG. 1 b). In contrast, 8.5 μg were necessary to induce acomparable toxic effect (FIG. 1 d). Thus, the calculatedefficacy/toxicity ratio of NGR-TNF under these conditions is 14 timesgreater than that of TNF.

Similar results were obtained with the B16F1 melanoma model. Treatmentwith 1 μg of NGR-TNF at day 11 and day 17, induced an anti-tumorresponse at day 19 greater than that obtained with 4 μg of TNF andsimilar to that obtained with 12 μg of TNF (data not shown). Incontrast, the loss of weight caused by 1 μg of NGR-TNF was markedlylower than that caused by 4 and 12 μg of TNF. Treatment with 12 μg ofNGR-TNF caused an even stronger anti-tumor effect, while the toxiceffect was similar to that of 12 μg of TNF.

When a third injection was done on day 19 some animal deaths occurred1-2 days later in all groups (2 out of 5 in the group treated withsaline and 12 μg of NGR-TNF and 1 out of 5 in the remaining groups). Ofnote, one animal treated with 12 μg of NGR-TNF completely rejected thetumor. When this animal was challenged with a second tumorigenic dose ofB16F1 cells, a palpable tumor developed after 18 days, while controlanimals developed a tumor within 6-7 days.

These results, altogether, suggest that the efficacy of NGR-TNF ininhibiting the tumor growth is 10-15 times greater than that of TNFwhereas the toxicity is similar. Moreover, NGR-TNF can induce protectiveimmune responses more efficiently than TNF.

EXAMPLE IV

Mechanism of Action of NGR-TNF

Anti-mouse CD13 mAb R3-63 purified from ascitic fluids by protein-GSepharose chromatography (Pharmacia-Upjohn, Uppsala, Sweden), anddialyzed against 0.9% sodium chloride.

Rabbit polyclonal antiserum was purchased from Primm srl (Milan, Italy)and purified by affinity chromatography on protein-A-Sepharose(Pharmacia-Upjohn). CNGRC (SEQ ID NO: 5) and CARAC (SEQ ID NO: 19)peptides were prepared as described previously (28).

To provide evidence that the improved activity of NGR-TNF is dependenton tumor targeting via the NGR moiety we have investigated whether thein vivo activity of NGR-TNF can be partially competed by CNGRC (SEQ IDNO: 5). To this end we have administered NGR-TNF (1 μg) to RMA-T tumorbearing mice, with or without a molar excess of CNGRC (SEQ ID NO: 5). Inparallel, other animals were treated with TNF (3 or 9 μg), again with orwithout CNGRC (SEQ ID NO: 5). As expected, CNGRC (SEQ ID NO: 5)decreased significantly the anti-tumor activity of NGR-TNF (FIG. 2 a)but not that of TNF (FIG. 2 b). At variance, a control peptide (CARAC)(SEQ ID NO: 19) was unable to cause significant decrease of NGR-TNFactivity (FIG. 2 c). These results suggest that CNGRC (SEQ ID NO: 5)competes for the binding of NGR-TNF to a CNGRC (SEQ ID NO: 5) receptor,and support the hypothesis of a targeting mechanism for the improvedactivity. Of note, CNGRC (SEQ ID NO: 5) was unable to decrease the invitro cytotoxic activity of NGR-TNF (data not shown).

Since it has been recently reported that aminopeptidase N (CD13) is areceptor for CNGRC (SEQ ID NO: 5) peptides, we then investigated thecontribute of this receptor in the targeting mechanism of NGR-TNF. Tothis end, we studied the effect of an anti-CD13 mAb (R3-63) on theanti-tumor activity of NGR-TNF and TNF. MAb R3-63 significantlyinhibited the anti-tumor activity of NGR-TNF (FIG. 2 a) but not that ofTNF (FIG. 2 b) indicating that CD13 is indeed critically involved in theanti-tumor activity of NGR-TNF. No expression of CD13 on RMA-T cellsurface was observed by FACS analysis of cultured cells with mAb R3-63(not shown), suggesting that other cells were recognized by the antibodyin vivo.

Although these data indicate that CD13 is an important receptor forNGR-TNF, we cannot entirely exclude that binding to other not yetidentified NGR receptors also contribute, albeit to a lower extent, tothe targeting mechanism.

Preliminary experiments of partial proteolysis showed that the Arg-Serbond in the N-terminal segment of TNF (residues 2-3) is very sensitiveto trypsin, whereas the rest of the molecule is much more resistant.Thus, to provide further evidence that the improved activity of NGR-TNFis related to its NGR moiety, we tried to cleave out the NGR domain fromthe N-terminal region of the mutein by partial digestion withimmobilized trypsin. This treatment converted both NGR-TNF and TNF intoa molecule corresponding to the TNF3-156 fragment (expected mass 16986.2Da; see FIG. 3 a for measured mass and expected sequences).

While digestion did not decrease the in vitro cytolytic activity ofNGR-TNF on L-M cells (2.3±1.4)×10⁸ U/mg) its in vivo anti-tumor activitywas decreased to the level of TNF (FIG. 3 b). Of note, the toxicity ofNGR-TNF and TNF were similar both before and after digestion, as judgedfrom animal weight loss one day after treatment (FIG. 3 b, right panel),suggesting that the NGR-dependent targeting mechanism does not altersthe toxicity.

EXAMPLE V

Preparation and Characterization of Human TNF and NGR-TNF

Human recombinant TNF and NGR-TNF (consisting of human TNF1-157 fusedwith the C terminus of CNGRCG (SEQ ID NO: 18)) were prepared byrecombinant DNA technology and purified essentially as described formurine TNF and NGR-TNF. The cDNA coding for human NGR-TNF was preparedby PCR on plasmid pET11b/hTNF containing the hTNF coding sequence (33),using the following primers: NGR-hTNF/1 (sense): 5′A TAT CAT ATG TGC AACGGC CGT TGC (SEQ ID NO:10) GGC GTC AGA TCA TCdT TCT CG 3′. NGR-hTNF/2(antisense): 5′ TCA GGA TCC TCA CAG GGC AAT GAT (SEQ ID NO:11) CCC AAAGTA GAC 3′.

The amplified fragment was digested and cloned in pET-11b (Nde I/BamH I)and used to transform BL21(DE3) E. coli cells (Novagen). The expressionof NGR-hTNF was induced with isopropyl-β-D-thiogalactoside, according tothe pET11b manufacturer's instruction. Soluble NGR-TNF was recoveredfrom two-liter cultures by bacterial sonication in 2 mMethylenediaminetetracetic acid, 20 mM Tris-HCl, pH 8.0, followed bycentrifugation (15000×g, 20 min, 4° C.).

The extract was mixed with ammonium sulfate (35% of saturation), leftfor 1 h at 4° C., and further centrifuged, as above. The ammoniumsulfate in the supernatants was then brought to 65% of saturation, leftat 4° C. for 24 h and further centrifuged. Each pellet was dissolved in1 M ammonium sulfate, 50 mM Tris-HCl, pH 8.0, and purified byhydrophobic interaction chromatography on Phenyl-Sepharose 6 Fast Flow(Pharmacia-Upjohn) (gradient elution, buffer A: 100 mM sodium phosphate,pH 8.0, containing 1 M ammonium sulfate; buffer B: 70% ethylen glycol,5% methanol, 100 mM sodium phosphate, pH 8.0). Fractions containing hTNFimmunoreactive material (by ELISA) were pooled, dialyzed against 20 mMTris-HCl, pH 8.0 and further purified by ion exchange chromatography onDEAE-Sepharose Fast Flow (Pharmacia-Upjohn) (gradient elution, buffer A:20 mM Tris-HCl, pH 8.0; buffer B: 1 M sodium chloride, 20 mM Tris-HCl,pH 8.0). All solutions employed in the chromatographic steps wereprepared with sterile and endotoxin-free water (Salf, Bergamo, Italy).

At this point about 30 mg of TNF and 32 mg NGR-TNF was recovered fromtwo-liters cultures. Non reducing SDS-PAGE showed bands corresponding tomonomers, dimers and trimers suggesting that also human NGR-TNF was amixture of trimers with correct intra-chain disulfides and trimers withone or more interchain disulfide bridges (FIG. 4A, lane b), as observedwith murine NGR-TNF.

Trimers with correct intrachain disulfide bridges were isolated fromthis mixture by a four-step denaturation-refolding process as follows:purified human NGR-TNF was denatured with 7 M urea and gelfilteredthrough an HR Sephacryl S-300 column (1025 ml) (Pharmacia)pre-equilibrated with 7 M urea, 100 mM Tris-HCl, pH 8.0. Fractionscorresponding to monomeric TNF were pooled, ultrafiltered through an YMMWCO 10 kDa membrane (Amicon) and refolded by dialysis against 33volumes of 2.33 M urea, 100 mM Tris-HCl, pH 8 at 4° C. (140 min)followed by 1.55 M urea, 100 mM Tris-HCl, pH 8 (140 min) and 1 M urea,100 mM Tris-HCl, pH 8 (140 min). Finally the product was dialyzedagainst 80 volumes of 100 mM Tris-HCl (16 h), centrifuged at 13000×g (30min), filtered through a SFCA 0.45 μm membrane (Nalgene) and gelfilteredthrough an HR Sephacryl S-300 column (1020 ml) pre-equilibrated with0.15 M sodium chloride, 0.05 M sodium phosphate (PBS). About 23 mg ofrefolded protein was recovered.

The final product was mostly monomeric after non reducing SDS-PAGE (FIG.4A, lane c), had an hydrodynamic volume similar to that of trimerichuman TNF by analytical gel-filtration HPLC on a Superdex 75 HR column(not shown), and had a molecular mass of 17937.8+1.8 Da (expected forCNGRCG-TNF1-157 (SEQ ID NO: 18), 17939.4 Da) by electrospray massspectrometry. The in vitro cytolytic activities of non-refolded andrefolded NGR-TNF on mouse L-M cells were (6.11×107)+4.9 and(5.09×107)+0.3 units/mg respectively, whereas that of purified human TNFwas (5.45×107)+3.1 units/mg. These results suggest that thedenaturation-refolding process did not affect the interaction of humanNGR-TNF with the murine p55 receptor.

The in vivo anti-tumor activity of 1 μg of human NGR-TNF (non refolded)was greater than that of 10 μg of TNF (FIG. 4B) whereas the toxicity, asjudged by animal weight loss, was significantly lower (FIG. 4C). Afterrefolding 0.3 μg of NGR-TNF was sufficient to induce an anti-tumoreffect stronger than that achieved with 10 μg of TNF (FIGS. 4D, 4E).

These results indicate that the anti-tumor activity of human NGR-TNF isgreater than that of human TNF.

Furthermore, we have observed that refolded human and mouse NGR-TNF caninduce significant anti-tumor effects on RMA-T-bearing mice even at verylow doses (1-10 ng/mouse) with no evidence of toxic effects, while TNFwas unable to induce significant effects at these doses (not shown).

EXAMPLE VI

Preparation and Characterization of Mouse NGR-IFNγ

Recombinant murine interferon (IFN)γ fused with CNGRCG (SEQ ID NO: 18)(NGR-IFNγ) was prepared by recombinant DNA technology, essentially asdescribed for NGR-TNF. The CNGRC (SEQ ID NO: 5) domain was fused withthe C terminus of IFNγ. Moreover the cysteine in position 134 wasreplaced with a serine; a methionine was introduced in position −1 forexpression in E. coli cells. The PCR primers used for the production ofthe NGR-IFNγ cDNA were: 5′-A TAT CTA CAT ATG CAC GGC ACA GTC ATT GAA AGCC (sense) (SEQ ID NO: 12) and 5′-TC GGA TCC TCA GCA ACG GCC GTT GCA GCCGGA GCG ACT CCT TTT CCG CTT CCT GAG GC (SEQ ID NO: 13). The cDNA wascloned cloned in pET-11b (Nde I/BamH I) and used to transform BL21(DE3)E. coli cells (Novagen). Protein expression was induced withisopropyl-β-D-thiogalactoside, according to the pET11b manufacturer'sinstruction. The product was purified from E. coli extracts byimmunoaffinity chromatography using an anti-mouse IFNγ mAb (AN18)immobilized on agarose, according to standard techniques. Reducing andnon reducing SDS-PAGE of the final product showed a single band of 16kDa. Electrospray mass spectrometry showed a molecular weight of16223+3.6 Da (expected, 1625.5 Da) corresponding to murineMet-IFNγ1-134(C134S)CNGRC (SEQ ID NO: 5) (NGR-IFNγ).

The capability of NGR-IFNγ and NGR-TNF to compete the binding of ananti-CD13 antibody to tumor associated vessels was investigated by usingan immunohistochemical approach.

Fresh surgical specimens of human renal cell carcinoma were obtainedfrom the Histopathology Department of the San Raffaele H ScientificInstitute. Sections (5-6 μm thick) of Bouin-fixed (4-6 h)paraffin-embedded specimens were prepared and adsorbed onpolylysine-coated slides. CD13 antigen were detected using theavidin-biotin complex method as follows: tissue sections were rehydratedusing xylenes and graded alcohol series, according to standardprocedures. Tissue sections were placed in a vessel containing 1 mM EDTAand boiled for 7 min using a micro-wave oven (1000 W). The vessel wasthen refilled with 1 mM EDTA and boiled again for 5 min. The tissuesections were left to cool and incubated in PBS containing 0.3% hydrogenperoxide for 15 min, to quench endogenous peroxidase. The samples werethen and rinsed with PBS and incubated with 100-200 μl of PBS-BSA (1 hat room temperature) followed by the mAb WM15 (anti-hCD13), alone ormixed with various competitor agents (see Table 2) in PBS-BSA (overnightat 4° C.). The slides were then washed 3 times (3 min each) with PBS andincubated with PBS-BSA containing 2% normal horse serum (PBS-BSA-NHS)(Vector Laboratories, Burlingame, Calif.) for 5 min. The solution wasthen replaced with 3 μg/ml biotinylated horse anti-mouse IgG (H+L)(Vector Laboratories, Burlingame, Calif.) in PBS-BSA-NHS and furtherincubated for 1 h at room temperature. The slides were washed again andincubated for 30 min with Vectastain Elite Reagent (Vector Laboratories,Burlingame, Calif.) diluted 1:100 in PBS. A tablet of3,3′-diamino-benzidine-tetrahydrocloride (Merck, Darmstadt, Germany) wasthen dissolved in 10 ml of deionized water containing 0.03% hydrogenperoxide, filtered through a 0.2 μm membrane and overlaid on tissuesections for 5-10 min. The slides were washed as above andcounterstained with Harris' hematoxylin. The tumor associated vesselswere identified by staining serial sections of the tissue with ananti-CD31 mAb (mAb JC/70A, anti-human CD31, IgG1, from DAKO, Copenhagen,Denmark).

The results are summarized in Table 2. As shown, the binding of WM15 totumor associated vessels was inhibited by an excess of NGR-TNF, NGR-IFNγand CNGRC (SEQ ID NO: 5), but not by other control reagents lacking theNGR motif. This suggests that the NGR binding site on CD13 stericallyoverlaps with the WM15 epitope. In contrast, NGR-TNF was unable tocompete the binding of 13C03 to epithelial cells.

We conclude that the NGR moiety of NGR-IFNγ and NGR-TNF and can interactwith a CD13 form recognized by mAb WM15 on tumor associated vessels.Moreover, these results indicate that the CNGRC (SEQ ID NO: 5) motif isfunctional either when linked to the N-terminus or the C-terminus of acytokine. TABLE 2 Binding of WM15 to renal cell cancer sections in thepresence of various competitors Binding of WM15 to tumor Competitorassociated vessels None + NGR-TNF (25 μg/ml) − NGR-IFNγ (50 μg/ml) −CNGRC (100 μg/ml) − TNF (25 μg/ml) + Human serum albumin (25 μg/ml) +Synthetic CgA(60-68) (100 μg/ml) +^(a)The competitor, in PBS containing 2% BSA, was added in the blockingstep and mixed with the primary antibody.^(b)mAb WM15 (anti-human CD13, IgG1) was from Pharmingen (San Diego,CA); the synthetic peptide CgA(60-68) corresponds to the chromogranin Afragment 60-68.

EXAMPLE VII

Targeted Delivery of Biotinylated NGR-TNF to Tumors Using Anti-TumorAntibodies and Avidin (Pre-Targeting)

The following example illustrates the possibility of “dual” targeting ofTNF, based on the combination of a tumor homing antibody and the peptideCNGRC (SEQ ID NO: 5).

A biotin-NGR-TNF conjugate was prepared by mixing NGR-TNF withD-biotinyl-6-aminocaproic acid N-hydroxysuccinimide ester (SocietaProdotti Antibiotici S.p.A, Milan, Italy), in 1 M sodium-carbonatebuffer, pH 6.8 (3 h at room temperature) (21). The reaction was blockedwith 1 M Tris-HCl, pH 7.5.

The conjugate was characterized by mass spectrometry and found tocontain 1 biotin/trimer (on average). C57BL/6 (Charles RiverLaboratories, Calco, Italy) were then challenged with 5×10⁴ RMA-T livingcells, s.c. in the left flank. When the tumor area reached 40 mm², micewere treated by sequential injections of biotinylated antibody, avidinsand biotin-TNF according to a “three-day” protocol as describedpreviously (26). We injected: 40 μg biotin-mAb19E12 (i.p., step I), 60μg avidin and 60 μg streptavidin after 18 and 19 h, respectively (i.p.,step II), 3 μg of biotin-NGR-TNF, 24 h later (i.p, step III). Eachcompound was diluted with a sterile 0.9% sodium chloride solution. Incontrol experiment, avidin and streptavidin were omitted. Eachexperiment was carried out with 5 mice/group. The tumor growth wasmonitored daily by measuring the tumor size with calipers. The tumorareas before and 10 days after treatment were 39±4 mm² and 8±5 mm²,respectively, in the group treated with mAb 19E12-biotin/avidin/streptavidin/biotin-NGR-TNF (5 animals, mean±SE). Inthe control group (treated with mAb 19E12-biotin/biotin-NGR-TNF alone)the tumor areas before and 10 days after treatment were 40±4 mm² and20±6 mm² respectively, indicating that pre-targeting with tumor homingantibody and avidin has increased the activity of NGR-TNF.

EXAMPLE VIII

Synergistic Activity Between NGR-TNF and Interferon-γ

Mouse B16F1 melanoma cells were cultured as described previously (Curniset al., 2000; Moro et al., 1997).

The neutralizing anti-IFNgamma monoclonal antibody AN18 was kindlysupplied by P.Dellabona, Milan, Italy). Doxorubicin (Adriblastina) waspurchased from Pharmacia-Upjohn (Milan, Italy). Recombinant murine IFNγwas purchased from Peprotech Inc. (USA) (endotoxin content: <1Units/μg). TS/A cells from a BALB/c spontaneous mammary adenocarcinomawere cultured in RPMI 1640 medium, 10% fetal bovine serum, 100 U/mlpenicillin, 100 μg/ml streptomycin, 0.25 μg/ml amphotericin B, 2 mMglutamine and 1% minimal essential medium with nonessential amino acids(BioWhittaker Europe, Verviers, Belgium).

Murine TNF and NGR-TNF (consisting of TNF fused with the C-terminus ofCNGRCG (SEQ ID NO: 18)) was prepared by recombinant DNA technology andpurified from E. coli cell extracts, as described (Curnis, et al.,2000). All solutions used in the chromatographic steps were preparedwith sterile and endotoxin-free water (Salf, Bergamo, Italy). Proteinconcentration was measured with a commercial protein quantificationassay kit (Pierce, Rockford, Ill.). The cytolytic activity of NGR-mTNFwas 9.1×10⁷ Units/mg. The hydrodynamic volume of NGR-mTNF was similar tothat of mTNF, a homotrimeric protein (Smith and Baglioni, 1987), by gelfiltration chromatography on a Superdex 75 HR column (Pharmacia,Sweden). Endotoxin content of NGR-mTNF was 0.082 Units/pg.

DNA manipulations were performed by standard recombinant DNA methods.The cDNA coding for murine IFNγ was prepared by PCR on cDNA obtainedfrom murine lymphocytes stimulated with phorbol 12-myristate 13-acetate,using the following primers: 5′AGAATTCATGAACGCTACACACTGCATCTTGGC 3′ (SEQID NO: 14) (forward primer); 5′TATATTAAGCTTTCAGCAGCGACTCCTTTTCCGC 3′(SEQ ID NO: 15) (reverse primer). Primers were designed to amplify thecDNA sequence coding for the full-length mouse IFNγ, including theleader sequence. They include the EcoRI and HindIII restriction site(underlined) for the cloning into the mammalian expression vectorpRS1-neo, to generate pRS1neo-IFNγ. pRS1neo-IFNγ was then prepared usingthe Plasmid Maxi Kit (Qiagen Inc.-Diagen, GmbH, Germany) and diluted at1 mg/ml in sterile and endotoxin-free water (S.A.L.F. LaboratorioFarmacologico SpA, Bergamo, Italy). pRS1neo-IFNγ (3 γg) was mixed with100 μl of 0.03 mg/ml Lipofectin Reagent (Gibco Brl) in RPMI 1640 andincubated for 20 min at room temperature. Then the mixture was added toTS/A cells plated in 24-well microtiter plates 1 day before (4×10⁴ cellsper well in 200 μl of culture medium). After incubation at 37° C., 5%CO₂ for 4 h, 2 ml of culture medium were added to each well. After 48 hof incubation the culture medium was changed with RPMI 1640 supplementedwith 10% fetal bovine serum, 2 mM glutamine and containing 1 mg/mlgeneticin. One week later, cells surviving selection were cloned bylimiting dilution in 96-well microtiter plates in the presence ofgeneticin. The supernatants of each clone was tested by IFNγ-ELISA. TenIFNγ-secreting clones were obtained. One clone, named TS/A-IFNγ,producing 1.13 μg IFNγ per ml of culture medium was selected and usedfor in vivo experiments.

IFNγ-ELISA. PVC microtiter plates (Becton Dickinson, cod.3912) werecoated with 5 μg/ml mAb AN18 in PBS (16 h at 4° C.). Plates were washedthree times with PBS and blocked with 2% bovine serum albumin (BSA) inPBS (PBS-BSA, 1 h at 37° C.). Plates were then washed three times withPBS and incubated with cell culture supernatants or mouse IFNγ standardsolutions diluted in PBS-BSA. Plates were washed eight times with PBScontaining 0.05% Tween-20 (Merck) (PBS-TW) and incubated with mAbXMG1.2-bio (0.2 μg/ml in PBS, 1 h at 37° C.). Plates were washed againwith PBS-TW and incubated for 1 h at 37° C. withstreptavidin-horseradish peroxidase (Sigma) diluted 1:3000 in PBS-BSA.After washing with PBS-TW, bound peroxidase was detected witho-phenylenediamine dihydrochloride peroxidase substrate (Sigma). Thechromogenic reaction was blocked after 30 min by adding 10% sulfuricacid. The A₄₉₀ was measured with an ELISA microplate reader (Biorad).

Studies on animal models were approved by the Ethical Committee of theSan Raffaele H Scientific Institute and performed according to theprescribed guidelines. C57BL/6 mice (Charles River Laboratories, Calco,Italy) weighing 16-18 g were challenged with subcutaneous injection inthe left flank of 5×10⁴ or B16F1 living cells; 5 days later, the micewere treated with NGR-mTNF and mIFNγ solutions (100 μl) followed 2 hlater by administration of doxorubicin solution (100 μl). All drugs wereadministered intraperitoneally (i.p). The drugs were diluted with 0.9%sodium chloride, containing 100 μg/ml endotoxin-free human serum albumin(Farma-Biagini, Lucca, Italy), except for doxorubicin, which was dilutedwith 0.9% sodium chloride alone. Tumor growth was monitored daily bymeasuring the tumors with calipers as previously described (Gasparri etal., 1999). Animals were sacrificed before the tumors reached 1.0-1.5 cmin diameter. Tumor sizes are shown as mean±SE (5 animals/group).

Endogenous IFN is Critical for NGR-TNF/Doxorubicin Therapeutic Activity.

We have shown previously that NGR-mTNF and doxorubicin play synergisticeffects in B16-F1 tumor-bearing immunocompetent mice, the anti-tumoractivity of doxorubinin being significantly increased bypre-administration of 0.1 ng of NGR-mTNF (above and Curnis et al.,2002). Accordingly, administration of low amounts of NGR-mTNF (0.1 ng)in combination with doxorubicin (80 μg) to B16-F10 tumor-bearing miceinduced anti-tumor effects stronger than those obtained with doxorubicinalone (FIG. 5). These drugs act synergistically, as NGR-TNF alone ispoorly active when used at low dosage (0.1 ng) (Curnis et al., 2002).

To assess the functional importance of endogenous mIFNγ in theanti-tumor activity of NGR-mTNF in immunocompetent mice, we studied theeffect of a neutralizing anti-mIFNγ antibody (mAb AN18) on theanti-tumor activity of NGR-mTNF/doxorubicin in C57BL6 mice.

When this antibody was administered 24 h before NGR-mTNF, thesynergistic effect between NGR-mTNF and doxorubicin was abolished (FIG.5), supporting the hypothesis that endogenous mIFNγ is an importantplayer in the anti-tumor activity of NGR-mTNF.

To further support the role of IFN in the therapeutic response to thesedrugs we performed other in vivo experiments using BALB/c IFNγ^(−/−)“knock out” mice, bearing subcutaneous mouse TS/A-mammary adenocarcinomatumors. In parallel, we performed similar experiments using wild-typeBALB/c IFNγ^(+/+) mice. NGR-TNF/doxorubicin significantly reduced thetumor mass in BALB/c IFNγ^(+/+) mice (FIG. 6, black bars), but not inBALB/c IFNγ^(−/−) mice (white bars). Notably, administration ofexogenous IFN (300 ng) in combination with NGR-TNF and doxorubicin toBALB/c IFNγ^(−/−) mice restored the synergy between these drugs (FIG. 6,white bars). These results confirm the hypothesis that IFN is necessaryfor the NGR-TNF/doxorubicin synergistic activity. Co-administration ofIFN and doxorubicin without NGR-TNF did not induce significantanti-tumor effects in BALB/c IFNγ^(−/−) mice (FIG. 6, white bars)indicating that this cytokine acts synergistically with NGR-TNF, andlittle or not with doxorubicin.

Role of T-Cells and Locally Produced IFN in NGR-TNF/DoxorubicinTherapeutic Activity.

T- and NK-cells are the primary sources of IFN in immunocompetent mice.To investigate the importance of T-cells as a source of the IFN in theNGR-TNF/doxorubicin combined therapy we investigated the effect of thesedrugs in B16F1 tumor-bearing nu/nu mice, lacking T-cells. In this modelthe NGR-TNF/doxorubicin synergistic activity was lost (FIG. 7A-C).However, when these drugs were administered in combination with IFN, thesynergistic effect was observed again (FIG. 7D). It is likely that theamount of endogenous IFN in these animals was not sufficient to activatethe NGR-TNF/doxorubicin synergism, while administration of exogenous IFNrestored the synergy.

The results of in vivo experiments with immunocompetent and nu/nu micemay suggest that T-cells present within the tumor mass ofimmunocompetent mice, but not of nu/nu mice, produce sufficient amountsof IFN to stimulate a synergistic response between NGR-TNF andchemotherapy. To assess whether the production of IFN within the tumormicroenvironment could restore the synergistic effect in nu/nu mice, wetransfected TS/A cells with murine IFN cDNA (FIG. 8). One clone able tosecrete IFN in culture medium was selected and named TS/A-IFN. TS/A-IFNand wild-type TS/A cells were then implanted, subcutaneously, in nu/numice. As expected, NGR-TNF/doxorubicin exerted significant anti-tumoreffects against TSA-IFN, but not against TS/A tumors, stronglysuggesting that locally produced IFN is indeed critical for theNGR-TNF/doxorubicin synergism.

In conclusion, these and the above results indicate that the therapeuticactivity of NGR-TNF/doxorubicin strongly depends on local production ofIFN, likely secreted by tumor-infiltrating lymphocytes.

Exogenous IFN Enhances the Therapeutic Activity of NGR-TNF/Doxorubicinin Immunocompetent Mice.

The therapeutic activity of NGR-TNF and doxorubicin, in combination withexogenous IFN, (300 ng) was then evaluated using C57B16 mice bearingB16F1 tumors. As shown in FIG. 9, the anti-tumor activity of the triplecombination was greater than that of NGR-TNF/doxorubicin. Thus,administration of exogenous IFN, together with NGR-TNF/doxorubicin,induced stronger anti-tumor effect also in immunocompetent mice.

Mechanism of Action of the Triple Combination (IFN, NGR-TNF andDoxorubicin).

We have shown previously that an important mechanism for theNGR-TNF/doxorubicin synergism is related to alteration of endothelialbarrier function by NGR-TNF and increased penetration of doxorubicin intumors. Thus, we investigated whether IFN is critical for this effect.To this aim we measured the effect of exogenous IFN on the penetrationof doxorubicin in TS/A tumors (FIG. 10). This experiment takes advantagefrom the fact doxorubicin is a fluorescent compound and that thefluorescence intensity of tumor cells, recovered from animals aftertreatment, is an indication of the amount of doxorubicin that haspenetrated tumors. The experiment was carried out in nu/nu mice toreduce the effect of endogenous IFN. When tumor-bearing mice weretreated with NGR-TNF and, 2 h later, with doxorubicin, no significantincrease of doxorubicin was found in tumor cells, compared to untreatedcontrols. However, administration of IFN in combination with NGR-TNFincreased the penetration of doxorubicin in tumors. This suggests thatIFN is critical for the TNF-induced penetration of chemotherapeuticdrugs.

The results of this study suggest that endogenous IFN is critical forthe NGR-TNF/doxorubicin synergistic activity and that exogenous mIFNγtogether with NGR-mTNF induce stronger anto-timour effect in bothimmunodeficient and immunocompetent mice. This view is supported by theabove observations that a) a neutralizing anti-IFN antibody markedlyinhibits the NGR-TNF/doxorubicin synergistic activity in immunocompetentmice and b) no synergism occurs in mice lacking the IFN gene (IFN−/−mice).

Given that T- and NK-cells are the primary sources of IFN inimmunocompetent mice and that athymic nu/nu mice lack T-cells, the aboveresults strongly suggest that T cells are the main source of the IFNnecessary for NGR-TNF/doxorubicin activity. In accord with this view isthe finding that exogenous or endogenous IFN (produced by tumor cellstransfected with IFN cDNA) restore the NGR-TNF/doxorubicin synergisticactivity in nu/nu mice.

These results point to a crucial role for IFN in tumor vasculartargeting with NGR-TNF and doxorubicin.

The lack of therapeutic effects of IFN in combination with doxorubicin(without NGR-TNF) suggest this cytokine synergizes with NGR-TNF, andlittle or not with doxorubicin. Considering that chemotherapeutic drugsmust cross the vessel wall and migrate through the interstitium to reachthe cancer cells, the site of action of IFN and NGR-TNF is very likelythe endothelial lining of vessels. It is well known that TNF can induce,in endothelial cells, alteration of cytoskeletal actin and formation ofintercellular gaps leading to increased permeability to macromolecules.On the same cells TNF can induce leukocyte adhesion molecules,proinflammatory cytokines, fibrin deposition, nitric oxide production,and apoptosis. Accordingly, we found that the in vitro permeability ofeA endothelial cell monolayers to horseradish peroxidase was enhanced byexposure to TNF in combination with IFN compared to TNF alone. We cannotexclude that, in view of the various effects that both TNF and IFN canexert on endothelial cells, alteration of vascular permeability in vivocould be also the consequence of indirect effects related to localrelease of other important inflammatory molecules that affectendothelial permeability.

Our findings may also have other important implications. Several studiesin animal models and in patients showed that TNF can selectively affectand damage tumor vessels but not vessels associated to normal tissues.Accordingly, the micro- and macrovasculature of tumors, but not ofnormal tissue, has been observed to be extensively damaged afterpatients were given isolated limb perfusion with TNF in combination withand melphalan. The molecular basis of this selectivity is unclear. Ithas been hypothesized that structural differences within tumor vesselsand/or the presence of tumor derived-“sensitizing factors” could beresponsible for the TNF vascular selectivity. Our results suggest localproduction of IFN, could be one of these sensitizing factors.

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the invention will be apparent to thoseskilled in the art without departing from the scope and spirit of theinvention. Although the invention has been described in connection withspecific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are apparent to those skilled inmolecular biology or related fields are intended to be within the scopeof the following claims.

REFERENCES

-   1. Carswell, E. A., et al., An endotoxin-induced serum factor that    causes necrosis of tumors. Proc. Natl. Acad. Sci. USA 1975.    72:3666-70.-   2. Helson, L., et al., Effect of tumor necrosis factor on cultured    human melanoma cells. Nature 1975. 258:731-732.-   3. Tracey, K. J., and A. Cerami. Tumor necrosis factor, other    cytokines and disease. Ann. Rev. Cell Biol, 1993. 9:317-43.-   4. Hoogenboom, H. R., et al., Construction and expression of    antibody-tumor necrosis factor fusion proteins. Mol. Immunol. 1991.    28:1027-37.-   5. Loetscher, H., et al., Human tumor necrosis factor alpha (TNF    alpha) mutants with exclusive specificity for the 55-kDa or 75 kDa    TNF receptors. J. Biol. Chem. 1993. 268:26350-7.-   6. Yang, J., et al., A genetically engineered single-chain FV/TNF    molecule possesses the anti-tumor immunoreactivity of FV as well as    the cytotoxic activity of tumor necrosis factor. Mol. Immunol. 1995.    32:873-81.-   7. Van Ostade, X., et al., Human TNF mutants with selective activity    on the p55 receptor. Nature 1993. 361:266-9.-   8. Paganelli, G., et al., Three-step monoclonal antibody tumor    targeting in carcinoembryonic antigenpositive patients. Cancer    Res. 1991. 51:5960-6.-   9. Paganelli, G., et al., Clinical application of the avidin-biotin    system for tumor targeting. In D. Goldenberg (Ed. Cancer therapy    with radiolabeled antibodies. CRC Press, Boca Raton, 1995. P.    239-253.-   10. Modorati, G., et al., Immunoscintigraphy with three step    monoclonal pretargeting technique in diagnosis of uveal melanoma:    preliminary results. Br. J. Ophtalm. 1994. 78:19-23.-   11. Colombo, P., et al., Immunoscintigraphy with anti-chromogranin A    antibodies in patients with endocrine/neuroendocrine tumors. J.    Endocr. Invest. 1993. 16:841-3.-   12. Debs, R. J., et al., Liposome-associated tumor necrosis factor    retains bioactivity in the presence of neutralizing anti-tumor    necrosis factor antibodies. J. Immunol. 1989. 143:1192-7.-   13. Debs, R. J., et al., Immunomodulatory and toxic effects of free    and liposome-encapsulated tumor necrosis factor alpha in rats.    Cancer Res. 1990. 50:375-80.-   14. Moro, M., et al., Tumor cell targeting with antibody-avidin    complexes and biotinylated tumor necrosis factor alpha. Cancer    Res. 1997. 57:1922-8.-   15. Schraffordt Koops, et al., Hyperthermic isolated limb perfusion    with tumour necrosis factor and melphalan as treatment of locally    advanced or recurrent soft tissue sarcomas of the extremities.    Radiothepray & Oncology 1998. 48:1-4.-   16. Lienard, D., et al., In transit metastases of malignant melanoma    treated by high dose rTNF alpha in combination with interferon-gamma    and melphalan in isolation perfusion. World Journal of Surgery 1992.    16:234-40.-   17. Hill, S., et al., Low-dose tumour necrosis factor alpha and    melphalan in hyperthermic isolated limb perfusion. Br. J.    Sugr. 1993. 80:995-7.-   18. Eggermont, A. M., et al., Isolated limb perfusion with tumor    necrosis factor and melphalan for limb salvage in 186 patients with    locally advanced soft tissue extremity sarcomas. The cumulative    multicenter European experience. Ann. Surg. 1996. 224:756-65.-   19. Mizuguchi, H., et al., Tumor necrosis factor alpha-mediated    tumor regression by the in vivo transfer of genes into the artery    that leads to tumor. Cancer Res. 1998. 58:5725-30.-   20. Pennica, D., et al., Cloning and expression in Escherichia coli    of the cDNA for murine tumor necrosis factor. Proc. Natl Acad. Sci.    USA 1985. 82:6060-4.-   21. Corti, A., et al., Tumor targeting with biotinylated tumor    necrosis factor alpha: Structure activity relationships and    mechanism of action on avidin pretargeted tumor cells. Cancer    Res. 1998. 58:3866-3872.-   22. Corti, A., et al., Up-regulation of p75 Tumor Necrosis Factor    alpha receptor in Mycobacterium avium-infected mice. Infect. Immun.    1999, 67:5762-5767.-   23. Corti, A., et al., Tumor necrosis factor (TNF) alpha    quantification by ELISA and bioassay: effects of TNF alpha-soluble    TNF receptor (p55) complex dissociation during assay incubations. J.    Immunol. Meth. 1994. 177:191-198.-   24. Ljunggren, H. G., and K. Karre. Host resistance directed    selectively against H-2-deficient lymphoma variants. Analysis of the    mechanism. J. Exp. Med. 1985. 162:1745-59.-   25. Celik, C., et al., Demonstration of immunogenicitywith the    poorly immunogenic B16 melanoma. Cancer Res. 1983. 43:3507-10.-   26. Gasparri, A., et al., Tumor pretargeting with avidin improves    the therapeutic index of biotinylated tumor necrosis factor alpha in    mouse models. Cancer Res. 1999. 59:2917-23.-   27. Palladino, M. A., Jr., et al., Characterization of the antitumor    activities of human tumor necrosis factor-alpha and the comparison    with other cytokines: induction of tumor-specific immunity. J.    Immunol. 1987. 138:4023-32.-   28. Arap, W., et al., Cancer treatment by targeted drug delivery to    tumor vasculature in a mouse model. Science 1998. 279:377-80.-   29. Fiers, W. Biologic therapy with TNF: preclinical studies. In V.    De Vita, S. Hellman, and S. Rosenberg Eds). Biologic therapy of    cancer: principles and practice. J. B. Lippincott Company,    Philadelphia, 1995. P. 295-327.-   30. Rathjen, D. A., et al., 1992. Selective enhancement of the    tumour necrotic activity of TNF alpha with monoclonal antibody.    Brit. J. Cancer 65:852.-   31. Robert, B., et al., 1996. Cytokine targeting in tumors using a    bispecific antibody directed against carcinoembryonic antigen and    tumor necrosis factor alpha. Cancer Res. 56:4758.-   32. Fraker, D. L., Alexander, H. R. & Pass, H. I., 1995. Biologic    therapy with TNF: systemic administration and isolation-perfusion.    In Biologic therapy of cancer: principles and practice, De Vita, V.,    Hellman, S. & Rosenberg, S. (eds) pp. 329-345. J. B. Lippincott    Company: Philadelphia.-   33. Pennica, D., et al., 1984. Human tumor necrosis factor:    precursor, structure, expression and homology to lymphotoxin.    Nature, 321, 724-729.-   34. Curnis, F., Sacchi, A., Borgna, L., Magni, F., Gasparri, A., and    Corti, A. (2000). Nat. Biotechnol. 18, 1185-90.-   35. Curnis, F., Sacchi, A., and Corti, A. (2002). Journal of    Clinical Investigation , In press.-   36. Gasparri, A., Moro, M., Curnis, F., Sacchi, A., Pagano, S.,    Veglia, F., Casorati, G., Siccardi, 36. A. G., Dellabona, P., and    Corti, A. (1999). Cancer Research 59,2917-23.-   37. Moro, M., Pelagi, M., Fulci, G., Paganelli, G., Dellabona, P.,    Casorati, G., Siccardi, A. G., and Corti, A. (1997). Cancer Research    57, 1922-8.-   38. Smith, R. A., and Baglioni, C. (1987). Journal of Biological    Chemistry 262, 6951-4.

1. A conjugation product between a cytokine selected from TNF or IFNγand a ligand of the CD13 receptor.
 2. A conjugation product as claimedin claim 1, wherein said cytokine is TNFα or TNFβ.
 3. A conjugationproduct as claimed in claim 1, wherein the ligand of the CD13 receptoris selected from the group consisting of antibodies or active fragmentsthereof, peptides or peptido-mimetics.
 4. A conjugation product asclaimed in claim 3, wherein said ligand is a peptide containing the NGRmotif.
 5. A conjugation product as claimed in claim 4, wherein saidpeptide is selected from the group consisting of CNGRCVSGCAGRC, NGRAHA,GNGRG, cycloCVLNGRMEC, linear CNGRC, and cyclic CNGRC.
 6. A conjugationproduct as claimed in claim 1, wherein the cytokine is derivatized withpolyethylene glycol or an acyl residue.
 7. A conjugation product asclaimed in claim 1, wherein the cytokine is further conjugated with acompound selected from the group consisting of an antibody, an antibodyfragment, and biotin, wherein said antibody or fragment thereof isdirected to a compound selected from the group consisting of a tumoralantigen, a tumoral angiogenic marker or a component of the extracellularmatrix.
 8. A conjugation product according to claim 7, wherein thecytokine is TNF and is conjugated to both a CD13 ligand and a compoundselected from the group consisting of an antibody, and antibodyfragment, and biotin.
 9. A cDNA encoding for a cytokine selected fromTNF and IFNγ bearing a 5′ or 3′ contiguous sequence encoding a CD13ligand.
 10. A cDNA according to claim 9, wherein said CD13 ligand is apeptide selected from the group consisting of CNGRCVSGCAGRC, NGRAHA,GNGRG, cycloCVLNGRMEC, linear CNGRC, and cyclic CNGRC.
 11. A vector forgene therapy containing the cDNA of claim
 9. 12. A pharmaceuticalcomposition comprising an effective amount of a conjugation product asclaimed in claim 1, together with pharmaceutically acceptable carriersand excipients.
 13. A composition as claimed in claim 12, in the form ofan injectable solution or suspension or a liquid for infusions.
 14. Acomposition as claimed in claim 12, in the form of liposomes.
 15. Apharmaceutical composition comprising an effective amount of aconjugation product of TNF and a ligand of the CD13 receptor or apolynucleotide encoding therefor, and an effective amount of IFNγ or apolynucleotide encoding therefor.
 16. A composition according to claim15 together with pharmaceutically acceptable carriers and excipients.17. A composition as claimed in claim 15, wherein said TNF is TNFα orTNFβ.
 18. A composition as claimed in claim 15, wherein the ligand ofthe CD13 receptor is selected from the group consisting of antibodies oractive fragments thereof, peptides or peptido-mimetics.
 19. Acomposition as claimed in claim 18, wherein said ligand is a peptidecontaining the NGR motif.
 20. A composition as claimed in claim 19,wherein said peptide is selected from the group consisting ofCNGRCVSGCAGRC, NGRAHA, GNGRG, cycloCVLNGRMEC, linear CNGRC, and cyclicCNGRC.
 21. A composition as claimed in claim 15, wherein the TNF isderivatized with polyethylene glycol or an acyl residue.
 22. Acomposition as claimed in claim 15, wherein the TNF is furtherconjugated with a compound selected from the group consisting of anantibody, an antibody fragment, and biotin, wherein said antibody orfragment thereof is directed to a compound selected from the groupconsisting of a tumoral antigen, a tumoral angiogenic marker or acomponent of the extracellular matrix.
 23. A composition according toclaim 22, wherein the TNF is conjugated to both a CD13 ligand and acompound selected from the group consisting of an antibody, and antibodyfragment, and biotin.
 24. A composition as claimed in claim 15, in theform of an injectable solution or suspension or a liquid for infusions.25. A composition as claimed in claim 15, in the form of liposomes. 26.A composition as claimed in claim 15, further comprising anotherantitumor agent or a diagnostic tumor-imaging compound.
 27. Acomposition as claimed in claim 26, wherein the another antitumor agentis doxorubicin.
 28. A method of treating or diagnosing a cancer patientcomprising administering the conjugation product of claim
 1. 29. Amethod of treating or diagnosing a cancer patient comprisingadministering the cDNA of claim
 9. 30. A method of treating ordiagnosing a cancer patient comprising administering the vector of claim11.
 31. A method of treating or diagnosing a cancer patient comprisingadministering the pharmaceutical composition of claim
 12. 32. A methodof treating or diagnosing a cancer patient comprising administering thepharmaceutical composition of claim
 15. 33. The method of claim 28comprising additionally administering other antitumor agents ordiagnostic tumor-imaging compounds.
 34. The method of claim 29comprising additionally administering other antitumor agents ordiagnostic tumor-imaging compounds.
 35. The method of claim 30comprising additionally administering other antitumor agents ordiagnostic tumor-imaging compounds.
 36. The method of claim 31comprising additionally administering other antitumor agents ordiagnostic tumor-imaging compounds.
 37. The method of claim 32comprising additionally administering other antitumor agents ordiagnostic tumor-imaging compounds.